Benzodioxole derivatives as modulators of proteolytic activity in plants

ABSTRACT

The present invention has as subject, the use of benzodioxole derivatives as modulators of the activity or of the content of protein hydrolases in plants. Such a new use allows the increase of the natural or induced defences of the plant.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 of PCTInternational application PCT/EP2003/001425, filed Feb. 13, 2003, whichwas published under PCT Article 21(2) in English.

FIELD OF THE INVENTION

The invention relates to the use of benzodioxole derivatives to modifyenzyme activity in plants.

STATE OF THE ART

Piperonyl butoxide is a benzodioxole polyoxyethylene known for some timeas a synergist of insecticides such as for example of pyrethrins,pyrethroids and carbamate types insecticides.

Its relatively low toxicity with regard to humans and animals and itsappreciable effects on a large spectrum of insecticides has allowed itsuse to spread rapidly in agriculture.

The mechanisms at the heart of this synergistic effect have not yet beenclarified, even if from time to time direct or indirect regulationeffects on some specific enzymes involved in the inactivation or in thecatabolism of the insecticide molecules with which it synergises havebeen proposed, such as for example non specific esterases present ininsect homogenates (Piperonyl Butoxide “The insecticide Synergist”,1998, Academic Press, p. 215), more recently on microsomal oxidases(Alzogaray R. A. Arch. Insect Biochem. Physiol., 2001, 46:119-126), oron cytochrome P450 mono oxygenases (Kotze et al. Int. J. Parasitol,1997, 27:33-40). However, as yet, an effect of PBO on enzymes of plantorigin has not been described, let alone in particular on theproteolytic or peptidase class of enzymes.

The significance of the proteases and their physiological inhibitors askey enzymes in the regulation of cellular processes both in the animaland plant kingdoms is known and widely recognised.

In plants, for example, the balance between proteolytic enzymes andnatural inhibitors is at the heart of the precise temporal regulationsof the germination process of dormant seeds.

It is also known that over the course of evolution, the production ofnatural inhibitors of proteolytic enzymes has been selected in plants,among others, as a protection mechanism against parasites. Theusefulness of interventions based on this type of approach is confirmedfor example in Harsulkar A. M. et al. Plant Physiol., 1999, 121:497-506.An approach based on the use of protease inhibitors expressedtransgenically, is described in EP 502730 and in EP 339009: in the firstthe expression of the inhibitor is sufficient to determine a protectiveeffect against nematodes, in the second, the transgenic expression of anatural protease inhibitor potentiates the insecticide effect of theproduct of the first gene encoding the Bt toxin (Bacillus thuringiensistoxin). The same approach is described by S. MacIntosh et al. in J.Agric. Food Chem. 1990, 38:1145-1152.

It is therefore evident that the availability of a product endowed withregulatory activity of the activity of proteolytic enzymes of plantorigin is of great industrial interest for a wide spectrum ofapplications.

SUMMARY OF THE INVENTION

The object of the present invention is the use of benzodioxolederivatives of formula I, amongst which the preferred is piperonylbutoxide (PBO) as modulators of the activity or of the content ofproteolytic enzymes in plants. Proteolytic enzymes able to hydrolysepeptide bonds, are preferably selected from the group consisting of:carboxypeptidases, aminopeptidases, dipeptidases, endopeptidases.

Treatment with benzodioxole derivatives is carried out on plants,preferably transgenic for a protein with insecticidic function,preferably belonging to the category of the Bacillus thuringiensistoxins (Bt-toxin), belonging to the Cry group. Preferably suchtransgenic plants are cotton, maize, tomato, potato and soya, or evenmore preferably, cotton.

According to a further aspect the invention extends to the use ofcompositions containing the benzodioxole derivatives as the activeingredients in combination with suitable emulsifiers and optionally withphotoprotective compounds selected from the group consisting of:benzotriazoles, benzophenones and sterically hindered amines, asmodulators of the activity or of the content of proteolytic enzymes inplants.

According to a further aspect the invention extends to a process forregulating the proteolytic activity in plants, preferably transgenic,even more preferably cotton, maize, soya, tomato, potato comprisingessentially the treatment of such plants with benzodioxole derivativesand with the compositions containing such compounds, in a way such thatthe final concentration of PBO is comprised of between 50 and 500grams/hectare and is performed at the end of the vegetative cycle.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Graphical representation of the inhibition of increasingconcentrations of PBO on the activity of the enzyme papain byDixon-plot. From the Dixon-plot obtained at two different substrateconcentrations (CBZ: L lysine-p-nitrophenyl ester), used at 1.5×10⁻⁴ M(full circle -●-) and 6×10⁻⁵ M (empty circle -◯-) respectively, it ispossible to calculate the inhibition constant (K_(I)) of PBO on theenzyme papain, which as equal to 2.6×10⁻⁴M. In addition, from thediagram it is possible to evaluate the IC₅₀ equal to 2×10⁻⁴M.

Abscissa: PBO concentration (M); ordinate: I/V (Δ Abs/min). Reversemicelles assay ISO-AOT 50 mM (AOT: double aerosol(2-ethylhexylsodiumsulfosuccinate in isooctane (ISO)); W_(o)(H₂O/AOT)=23

FIG. 2. Graphical representation of the inhibition of increasingconcentrations of PBO on the activity of the enzyme ficin by Dixon-plot.

From the Dixon diagram obtained at two different substrateconcentrations (CBZ: L lysine-p-nitrophenyl ester), used at 1.5×10⁻⁴ M(full circle -●-) and 6×10⁻⁵ M (empty circle -◯-) respectively, it ispossible to calculate the inhibition constant (K_(I)) of PBO on theenzyme ficin, equal to 0.45×10⁻³M. From the diagram it is also possibleto evaluate the IC₅₀ equal to 0.8×10⁻³M.

Abscissa: PBO concentration (M), ordinate: I/V (Δ Abs/min). Reversemicelles assay of ISO-AOT 50 mM (AOT: twin aerosol(2-ethylhexylsodiumsulfosuccinate in isooctane (ISO)); W_(o)(H₂O/AOT)=25

FIG. 3. Graphical representation of the inhibition of increasingconcentrations of PBO on the activity of the enzyme bromelain byDixon-plot.

From the Dixon-plot obtained at two different substrate concentrations(CBZ: L lysine-p-nitrophenyl ester), used at 1.5×10⁻⁴ M (full circle-●-) and 6×10⁻⁵ M (empty circle -◯-) respectively, it is possible tocalculate the inhibition constant (K_(I)) of PBO on the enzymebromelain, equal to 0.1×10⁻³M. In addition from the plot it is possibleto evaluate the IC₅₀ equal to 0.4×10⁻³M.

Abscissa: PBO concentration (M), ordinate: I/V (Δ Abs/min). Reversemicelles assay of ISO-AOT 50 mM (AOT: twin aerosol(2-ethylhexylsodiumsulfosuccinate in isooctane (ISO)); W_(o)(H₂O/AOT)=28

FIG. 4. Carboxypeptidase activity in cotton sprouts after 4 day ofgermination.

The extent of proteolytic activity in cotton sprouts was measured at30′, 60′, 90′ and 1.20° after suspension of the acetonic powder inaqueous buffer at pH 6.5 by determining the absorbance at 280 nm in aquartz cuvette, after protein precipitation with TCA and removal bycentrifugation.

Each series of analysis was performed in duplicate. The comparisonbetween the values of carboxypeptidase activity of treated and untreatedsamples was done using the average value of each series of analysis.

Treated samples: dark grey

Untreated samples: light grey

FIG. 5. Endopeptidase activity in cotton sprouts after 4 day ofgermination.

The extent of hydrolytic activity in cotton sprouts was measured at 15′,30′, 60′, 90′ and 120′ after solubilization of the acetonic powder inaqueous buffer at pH 7.7 by determining the absorbance at 280 nm in aquartz cuvette after protein precipitation with TCA and removal bycentrifugation. Each series of analysis was performed in duplicate. Thecomparison between the values of carboxypeptidase activity in treated(dark grey) and untreated (light grey) samples was done using theaverage value of each series of analysis. Abscissa: time (min);ordinate: Abs 280 nm.

FIG. 6. Bt content in PBO treated vs PBO untreated cotton plants.

An immunoenzymatic assay was used to determine the Bt levels atdifferent stages of plant development.

-   a) Measured Bt toxin values; ordinate: Bt expressed as ppm;    abscissa: days of plant culture and where different, plant tissue.    Black col.: Untreated cotton; grey col: PBO treated cotton. Standard    deviation is also indicated.-   b) % differences in Bt content during time in PBO treated vs    untreated plants. Ordinate: % Bt content (ppm) in PBO treated minus    untreated plants; abscissa: days of plant culture and where    different, plant tissue (cotyledons or leaves).

FIG. 7. Comparison between endopeptidase activity of treated with PBOand untreated cotton plants at different growing stages by the RadialDiffusion Assay.

The radium of the clear zone produced by the plant extracts was comparedto a standard curve obtained by a serial dilution of trypsin. A standardsolution of trypsin was used to plot a standard curve for each sampleplate, thus the endopeptidase activity was expressed as units of trypsinequivalents. For each extract, the amount of soluble proteins in mg wasdetermined by the Biuret method and the enzymatic activity was thenexpressed as specific activity i.e. Units/mg soluble proteins. Abscissa:time (days); ordinate: trypsin units/mg soluble protein.

FIG. 8. Comparison between peptidase activity (U/mg of soluble protein)of PBO treated and untreated cotton plants by a photometric assay withDL-BAPA as a substrate.

The photometric assay was carried out following the DL-BAPA hydrolysisat 410 nm. One peptidase unit (U) was defined as the amount of theenzyme, which produces one unit of absorbance variation at 410 nm/minuteat pH 8.2 and 30° C. For each extract, the amount of soluble proteins inmg was determined by the Biuret method and the enzymatic activity wasthen expressed as specific activity i.e. Units/mg soluble proteins.

Each analysis was performed in duplicate or in triplicate and thestandard deviation for each point is also indicated. Abscissa: time(days); ordinate: trypsin units/mg soluble protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of benzodioxole derivativescomprised in the following general formula I:

wherein R₁, R₂ and R₃ being the same or different are selected from thegroup consisting of: hydrogen; alkyl C₂-C₈; CH₂OR₄ where R₄ is selectedfrom the group consisting of: hydrogen, —(CH₂CH₂O)_(n)—R₅, in which n isan integer from 1 to 4 and R₅ is selected from the group consisting of:hydrogen, alkyl C₁-C₈, aryl non substituted or substituted by: alkylC₁-C₄, halogen, cyano group (CN), —SO₃H group, carboxyalkyl group—COOR₆, (where R₆ is hydrogen or alkyl C₁-C₈), a —N(R₇)—R₈ group, whereR₇ and R₈ being the same or different are: hydrogen or alkyl C₁-C₄ ortogether with the atom of nitrogen to which they are bound, representinga piperidinyl, pyrrolidine, morpholine group;R₅ is in addition selected from the group consisting of aralkyl C₇-C₉non substituted or substituted on the aromatic ring by substituentsselected from the group consisting of: alkyl C₁-C₄, halogen, a cyanogroup, a —SO₃H group, a carboxyalkyl group —COOR_(S), where R₉ has thesame meaning as R₆ and where, when R₁, R₂ and R₃ are the same, theycannot be hydrogen,said use based on the surprising finding that the previously definedcompounds are endowed with the capacity to modulate the activity or thecontent of plant proteolytic enzymes.

Preferably, in the above mentioned compounds, the substituents R₁, R₂and R₃ being the same or different are selected from the groupconsisting of: hydrogen, alkyl C₂-C₄, CH₂OR₄ where R₄ is selected fromthe group consisting of: hydrogen, —(CH₂CH₂O)_(n)—R₅, in which n is aninteger from 1 to 2 and R₅ is selected from the group consisting of:hydrogen, alkyl C₁-C₄, benzyl, aryl non substituted or substituted by:alkyl C₁-C₃, and where, when R₁, R₂ and R₃ are the same, they can neverbe hydrogen.

Still more preferably in such compounds of formula I, the substituentsR₁, R₂ and R₃ being the same or different, are selected from the groupconsisting of: hydrogen, propyl, CH₂OR₄ where R₄ is —(CH₂CH₂O)₂—R₅ andR₅ is selected from the group consisting of: alkyl C₂-C₄, phenyl,toluoyl and where, when R₁, R₂ and R₃ are the same, they can never behydrogen. Still more preferably such compounds correspond topiperonyl-butoxide (PBO) or5-[[2-(2-Butoxyethoxy)ethoxy]methyl]-6-propyl-1,3-benzodioxole, offormula (II):

For simplicity we will make reference in the following text, only to thecompound piperonyl-butoxide known by the abbreviation PBO or the term“benzodioxole derivatives”, being understood that with this abbreviationand with this term is intended to refer in the present application, toall the compounds of general formula I, comprising the preferredsubstituents.

For the purpose of the present invention the terms proteases,proteinases or peptidases are used in an equivalent manner and areintended to refer to the peptidic hydrolases, denominated for simplicityproteolytic enzymes or proteases over the course of the presentdescription, i.e. to enzymes with hydrolytic activity towards peptide oramidic bonds independently of their position, therefore either when theyare internal to the polypeptide chain, or at the N- or C-terminal ends.According to this definition therefore, both endopeptidases typeenzymes, and exopeptidases, such as the aminopeptidases orcarboxypeptidases which hydrolyse the peptide bonds liberating singleamino acids sequentially from the N- or C-terminal ends are comprisedwithin the definition of proteolytic enzymes.

The proteolytic enzymes on which PBO exherts its regulatory activity,are preferably selected from the group consisting of: carboxypeptidases,aminopeptidases, dipeptidases, endopeptidases, wherein theendopeptidases are preferably selected from the group consisting of:serine proteases, cystein proteases, cathepsins, metallo-endopeptidases;the cystein proteases are preferably selected from the group consistingof: bromelain, calpain, ficin, papain, chymopapain.

The regulation of proteolytic activity in plants, for example throughthe activation of specific inhibitors has a predominantly defensive rolein comparison to the proteases of insects and pathogenicmicro-organisms. In the case of lesions produced by mechanical orbiological means, protease inhibitors are synthesised de novocontributing to the defence strategy of the plant.

The modulatory potential of the inhibitors on the endogenous proteasescould be modest in seeds and tends to disappear during germination; animportant role has been attributed to the inhibitors during seedmaturation to prevent protein degradation during the accumulation phase.Therefore according to further object, the invention comprises as afurther embodiment the PBO proteolytic modulation activity on seeds;according to a further embodiment PBO is also useful to determine theactivation of plant's defensive pathways in the case of wounds orlesions allowing the regulation of general tissue growth.

A further advantage of the novel activity on plant cell proteolyticactivity herein described is the regulation of the production, of thematuration or of the degradation, or in other words of the turn-over ofendogenous proteinaceous substances, for example those with naturalinsecticide or fungicide functions or with tissue repair functions. Thismechanism may help in potentiating the natural response of the plantcell towards possible parasitic aggression, or towards externallyderived stresses.

An example of substances endowed with fungicide activity, is describedin Leah et al. J. Biol. Chem., 1991, 266:1564-1573 and is nonextensively enlisted herein: Ribosome Inactivating Proteins (RIP), whichhave specificity for only distantly correlated ribosomes, such as fungi,but not for plant ribosomes, or the chitinases and the(1-3)-β-glucanases, which interfere with the synthesis of the cell wallsof the fungus. Other substances produced by the plant in the form ofinactive protein precursors, and having defensive functions againstbacteria and fungi in their mature forms, are thionines, described forexample in Bohlmann H. Critical Reviews in Plant Sciences, 1994,13:1-16.

Treatment with PBO according to the novel use herein described iscarried out as known to the skilled man on alt plant types,concentrating on the air exposed areas of the plant, and in particularon the leaves. The treatment can also be performed on seeds. In itspreferred embodiment the treatment is preferably carried out ontransgenic plants selected from the group consisting of: cotton, maize,potato, tomato and soya. According to this preferred embodiment, theinvention refers to the use of benzodioxole derivatives as modulators ofthe proteolytic activity in plants transgenic for the insertion of atransgene encoding a protein.

The plant proteolytic activity variation obtained after treatment withPBO, has a differential effect depending on the system considered as itmay allows to increase or even to reduce the availability of a protein,or of a protein in its active conformation. It is known that a steadystate level is the result of the rate of protein degradation andproduction. However proteolysis is also known as a mechanism for proteinactivation. As a result, the new use allows an increase in the naturalor induced defences of the plant towards parasitic infection or externalattacks. Accordingly, a further and preferred embodiment of theinvention is the regulation of transgenically expressed protein levelsthrough the modulation of the proteolytic activity within a plant cell.Particularly preferred are plants transgenic for one of the Bacillusthuringiensis toxins.

In particular, in the case of plants transgenic for the Bacillusthuringiensis Bt toxin gene, the possibility of controlling themechanism of proteolysis is extremely important both to control theactivity of the transgenic toxin or to regulate its production.

It is however to be noted that this preferred embodiment of theinvention is not limited to a single production or activation mechanismon the transgenic protein, but extends to all the mechanisms activatedby PBO through a direct or indirect effect (such as through proteaseinhibitors) on proteolytic enzymes. An increase in the levels ofproteolytic enzymes can be monitored by direct or indirect assays. Amongthe indirect assays the activity of proteases on various endogenous orexogenous substrates can be measured according to methods well known inthe art.

In the case of transgenic plants, the transgene encodes one of the Cryprotein of Bacillus thuringiensis and even more preferably for a proteinselected from the group consisting of: CryI, Cry II, Cry III, Cry IV, inparticular CryIA (a), (b) or (c). According to a particularly preferredembodiment the plant is cotton and the transgene encodes for the CryItoxin of Bacillus thuringiensis.

The authors of the present invention have additionally observed that incotton transgenic for the Bt CryI protein, the amount of transgenictoxin after PBO treatment is higher than in transgenic untreated plantsand this finding correlates with a loss in proteolytic activity inPBO-treated plants versus untreated, during a period of at least 100days.

Hence the use of benzodioxole derivatives of formula I as proteolyticmodulators is particularly advantageous in transgenic plants, preferablyselected from the group consisting of: cotton, maize, tomato, potato orsoya preferably when they are transgenic for Bt-toxin, still morepreferably for the Cry I toxin, for inhibiting the proteolyticmechanisms directly or indirectly modifying the expression of thetransgenic (i.e. inactivating or reducing) Bt toxin in the plant cell.Particularly preferred is the PBO treatment of cotton transgenic for oneof the Bacillus thuringiensis toxins.

The levels of Bt toxin in plants are measured as known in the field, forexample with immunoenzymatic assays carried out with antibodies specificfor the Bt toxin. Alternatively, quantities of Bt toxin less than theuseful limits can be estimated by directly measuring the lack ofmortality in the parasitic insects in the field or in the laboratory.

According to an additional embodiment, the invention provides a processto regulate the proteolytic activity in plants, preferably to inhibit atleast partially the proteolytic activity of a plant, comprisingessentially the treatment of the plants with PBO or its derivatives orwith the compositions comprising PBO as the active ingredient, in a waysuch that the concentration of the active ingredient is comprised from50 to 800 grams/hectare, more preferably from 100 to 400 grams/hectare,even more preferably from 200 to 350 grams/hectare. The processaccording to the invention is preferably repeated up to three times pervegetative cycle and even more preferably is carried out at the end ofthe vegetative cycle. This preferred aspect is of particular relevancewhen the plant is transgenic and in particular when such plant,preferably cotton, maize, tomato, potato and soya, is transgenic for theCry toxin of Bacillus thuringiensis. As a matter of fact the modulatoryactivity of PBO is observed few hours after the treatment up to fewdays, during different phases of the plant growth cycle.

The modulation of protease activity by PBO in plants is preferably anegative modulation, through a direct inhibition of PBO on the enzyme,or through an indirect effect such as, for example, through theactivation of protease inhibitors or the de novo synthesis of specificprotease inhibitors.

Treatment with PBO is conveniently carried out using compositions withappropriate excipients or emulsifiers. Therefore according to a furtheraspect the invention relates to the use of compositions containing thebenzodioxole derivatives of formula I as the active ingredient, or thepreferred embodiments thereof, such as PBO, in combination withappropriate emulsifiers or excipients and optionally with photoprotectorcompounds, as modulators of the activity or the content of proteolyticenzymes in plants. The emulsifiers used are selected from the groupconsisting of: calcium salts of alkylarylsulphonic acids, polyglycolesters of fatty acids, alkylarylpolyglycol ethers, polyglycol ethers offatty alcohols, ethylene oxide-propylene oxide condensation products,alkyl polyethers, sorbitanic fatty acid esters, polyoxyethylensorbitanicfatty acid esters, polyoxyethylene sorbitanic esters. Particularlypreferred are the emulsifiers selected from the group consisting of:alkylarylpolyglycol ethers and calcium salts of alkylarylsulfonic acid.The emulsifiers are present at a minimum concentration of 2% (w/w).

The concentration of PBO in the compositions with emulsifiers orexcipients is comprised from 1 to 98% (w/w), preferably from 20 to 95%,even more preferably from 50 to 90%.

For the purposes herein the mixture of active ingredients in combinationwith the appropriate excipients or emulsifiers is denominated“concentrate”. To the concentrate is optionally added a protective agentagainst photo oxidation (photoprotectors) selected from the class ofcompounds consisting of: benzotriazoles, benzophenones, and stericallyhindered amines, in concentrations comprised from 0.1% to 10%,preferably from 0.5% to 8%, even more preferably from 1% to 5% in weightof the concentrate.

Amongst benzotriazoles, compounds are selected from the group consistingof: 2-(2′-hydroxy-5-t-octylphenyl)benzotriazole and2-(2′-hydroxy-3′,5′-di-t-butylphenyl)-5-chlorobenzotriazole.

Amongst benzophenones, compounds are selected from the group consistingof: 2-hydroxy-4-methoxy benzophenone, 2-hydroxy-4-octyloxy benzophenone,2′-dihydroxy-4,4′-dimethoxybenzophenone.

Amongst sterically hindered amines, compounds are selected from thegroup consisting of: di(2,2,6,6-tetramethyl-4-piperinidyl) sebacate;di(1,2,2,6,6-pentamethyl-4-piperidinyl) sebacate;alpha-[[6-[[4,6-bis(dibutylamino)-1,3,5-triazin-2-yl](2,2,6,6-tetramethyl-4-piperidinyl)amino]hexyl](2,2,6,6-tetramethyl-4-piperidinyl)amino]-omega-[4,6-bis(dibutylamino)-1,3,5-triazin-2-yl]-poly[[6-[butyl(2,2,6,6-tetramethyl-4-piperidinyl)amino]-1,3,5-triazin-2,4-diyl][2,2,6,6-tetramethyl-4-piperidinyl)imino]-1,6-hexandiyl[2,2,6,6-tetramethyl-4-piperidinyl)imino];polymer of dimethylsuccinate with4-hydroxy-2,2,6,6-tetramethyl-1-piperidinethanol; polymer of N,N′di(2,2,6,6-tetramethyl-4-piperinidyl)-1,6-hexanediamine with 2,4,6trichloro-1,3,5-triazine and 1,1,3,3-tetramethylbutylamine;poly-methylpropyl-3-oxy(4((2,2,6,6-tetramethyl)piperidinyl siloxane;1,3,5-triazine-2,4,6,-triamine, N,N′″[1,2-ethanediyl di[[[4,6-bis[butyl(1,2,2,6,6-pentamethyl-4-piperidinyl)amino]-1,3,5-triazine-2il]imino]-3,1-propanodiyl]]-di[N′,N″-dibutyl N′,N″-bis(1,2,2,6,6-pentamethyl-4-piperidinyle)]or the following mixture: mixture of the polymer of dimethylsuccinatewith 4-hydroxy-2,2,6,6-tetramethyl-1-piperidin ethanol and the polymerof N,N′ di(2,2,6,6-tetramethyl-4-piperinidyl)-1,6-hexanediamine with2,4,6 trichloro-1,3,5-triazine and 1,1,3,3-tetramethylbutylamine.

Particularly preferred are benzophenones, even more preferably2-hydroxy-4-methoxy benzophenone and 2-hydroxy-4-octyloxy benzofenone,and amongst sterically hindered amines, preferred compounds aredi(2,2,6,6-tetramethyl-4-piperinidyl) sebacate anddi(1,2,2,6,6-pentamethyl-4-piperidinyl) sebacate.

The concentrate, optionally containing the photoprotective agent, isemulsionable and is therefore mixed with water so as to obtain theappropriate solutions to be nebulised preferably by field spraying suchthat the concentration of PBO is comprised from 50 to 800 grams/hectare,preferably from 100 to 400 grams/hectare, even more preferably from 200to 350 grams/hectare. Every treatment cycle in the field can be composedof one up to three treatments per vegetative cycle.

The invention will be now better detailed in the following experimentalexamples, which do not represent any limitation thereof.

EXPERIMENTAL PART Example 1 In Vitro Inhibition of CysteinicEndopeptidases by Piperonyl Butoxide

The assays were carried out in the reverse-micelles assay dispersed inorganic solvent, described by Walde et al. in Eur. J. Biochem., 1988,173:401-409, and already reported in the literature in kinetic studiesof the inhibition of trypsin with natural and synthetic inhibitors. Suchan assay was adapted to the following purified plant enzymes: papain,ficin and bromelain (Sigma catalogue No: P4762, F4125, B5144respectively) in the presence of substrate CBZ (L lysine-p-nitrophenylester, Sigma catalogue No C3637), and carried out respectively with Wo(molar ratio water/surfactant or H₂O/AOT)=23, 25, 28.

The enzyme assay on reverse-micelle has been already described in theliterature for the enzyme trypsin and was adapted to different enzymes.These values were obtained by vigorously mixing, in a quartzspectrophotometry cell, 1 ml of 50 mM AOT-ISO with appropriate volumesof protease and different buffer solutions, respectively MES(2-[N-morpholino]ethanesulfonic acid) for the enzyme papain, HEPES(N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) for the enzymeficin and acetate for the enzyme bromelain. When the solution wascompletely transparent and apparently homogeneous, it was adjustedthermostatically to 30° C. To this solution was added an appropriatevolume of substrate CBZ (dissolved in acetonitrile/H₂O, 80:20 (v/v) at aconcentration of 15 mM) and following further agitation (the solutionreturned to being transparent), the change in absorbance was measured(Dabs) at 340 nm with a Cary 219 spectrophotometer. The assay conditionsin summary, were the following:

Papain: AOT-ISO: 50 mM (bis 2-etylhexyl sodium sulfosuccinate-isooctane) Wo: 23 buffer: 50 mM MES pH 6.2 containing 2.5 mM cystein,temperature: 30° C. substrate: 1.5 × 10⁻⁴M and 6 × 10⁻⁵M CBZ (Llysine-p-nitrophenyl ester, Sigma) papain: 2 mg/ml PBO (when present):from 4.87 × 10⁻⁴M to 5.9 × 10⁻³M Ficin: AOT-ISO: 50 mM (bis2-ethylhexylsodium sulfosuccinate- isooctane) Wo: 25 buffer: 50 mM HEPES pH 7.1containing 2.5 mM L-cystein, temperature: 30° C. substrate: 1.5 × 10⁻⁴ Mand 6 × 10⁻⁵M CBZ (L lysine-p-nitrophenyl ester, Sigma) ficin: 1.7 mg/mlPBO (when present): from 4.87 × 10⁻⁴M to 5.9 × 10⁻³M Bromelain: AOT-ISO:50 mM (bis2-ethylhexyl sodium sulfosuccinate- isooctane) Wo: 28 buffer:10 mM Acetate, pH 4.6 containing 1 mM L-cystein temperature: 30° C.substrate: 1.5 × 10⁻⁴M and 6 × 10⁻⁵M CBZ (L lysine-p-nitrophenyl ester,Sigma) bromelain: 2 mg/ml PBO (when present): from 4.87 × 10⁻⁴M to 5.9 ×10⁻³M.

The experimental data obtained are represented graphically in the formof Dixon-plot (described for example in: “Quantitative Problems inBiochemistry”, E A. Dawes, 5^(th) ed. 1972 Baltimore, The Williams andWilkins Company), in the FIGS. 1, 2 and 3. Such a graphicalrepresentation allows the calculation of the Ki values (inhibitionconstant) obtained for PBO respectively on papain: 2.6×10⁻³ M, on ficin:4.5×10⁻⁴ M and on bromelain: 1×10⁻⁴ M, therefore indicating that theinhibitory effect of PBO is higher on bromelain. The test results,obtained at non saturating substrate concentrations, indicate that,under these conditions, PBO inhibits all the proteases tested. Inparticular from the Dixon plots presented in FIGS. 1-3 it is possible toevaluate the IC₅₀ of PBO which on the papain system is equal to 2×10⁻³M, on the enzyme ficin it is equal to 0.8×10⁻³ M and on the enzymebromelain is equal to 0.4×10⁻³M.

Example 2 Inhibition of Bromelain by PBO

The assay was carried out as described in Heinrikson & Kezdy, 1976,Methods in Enzymol. 45, p. 740. The conditions are summarised brieflyherein: 50 μg of bromelain was mixed with variable quantities of NaCBZ-L-lysine p-nitrophenyl ester substrate in 3 ml of 10 mM acetatebuffer pH 4.6 containing KCl (0.1 M) and L-cystein (1 mM). The changesin the initial velocities of the reaction were measured by the changesin absorbance at 340 nm/min, in the presence of a 1% solution of PBO inH₂O in two different experiments.

The results are presented in table 1.

TABLE 1 The effect of 1% PBO on the activity of bromelain. Substrateconc. control test 1 test 2 Mean (% contr.) 24 μM 0.0199 0.0141 0.01300.0136 (68%) 48 μM 0.0297 0.0243 0.0297 0.0270 (68%) 96 μM 0.0623 0.05180.0461 0.0490 (78%)

The data indicate that 1% PBO has an inhibitory effect on bromelainwhich for substrate concentrations comprised of between 20-24 μM and48-50 μM, is variable from 65 to 80%.

The experiment was repeated using increasing concentrations of PBO and afixed substrate concentration (240 μM). The data are reported in table2.

TABLE 2 Effect of increasing quantities of PBO on the activity of theenzyme bromelain % PBO Exp. 1 Exp. 2 Exp. 3 (w/w) Initial velocity (% ofcontrol) 0 0.1382 0.1221 0.1051 0.334 0.1306 (95%) 0.1197 (98%) 0.10290.5 0.1252 (91%) — — 0.667 — 0.1079 (88%) — 1 0.1194 (86%) 0.1057 (86%)— 1.334 — 0.1029 (84%) — 1.667 — 0.0927 (76%) —

From the data in table 2 it may be observed that PBO at concentrationvalues higher than 0.5% has an inhibitory activity of the proteolyticenzyme bromelain. This effect is concentration dependent. The degree ofthe inhibition is in agreement with the data obtained in the previousexamples.

Example 3 Effect of PBO on Carboxypeptidase and Endopeptidase in CottonSeedlings

With the aim to evaluate the effect of PBO on the endogenous plantenzymatic system in cotton in vitro assays were carried out usingacetonic powders got from treated (PBO) and untreated freeze dried seedsprouts, without the addition of any specific exogenous substrate. Thebasic idea was to “freeze” the physiologic protein hydrolysis enzymaticactivity and to re-establish the process simply solubilizing theproteolytic enzymatic system in suitable buffers, which were 0.1 Mphosphate pH 6.5 and 0.1 M tris-HCl pH 7.7, for evaluating both thecarboxypeptidase and the endopeptidase activities according to themethod described by Hile et al. 1972; Funkhouser et al. 1980.

Materials and Methods

Cotton seeds (cv. Carmela) were obtained from “Semillas Battle”,Barcelona (E).

Germination Conditions

The seeds were previously rinsed in tap water and then imbibed withaeration in distilled water over night (Funkhouser, E. A. et al., Z.Pflanzenphysiol., 1980, 100, 319-324).

After this process, seeds were planted in settled and sterile sand andthen watered with distilled water (untreated seeds) and with the samevolume of a micro-suspension of saturated PBO (1% w/v) (treated seeds).The plastic boxes containing seeds were covered by a plastic film andplaced in a germinator in the dark for 4 days at 30° C. as described inHile, J. N. and Dure, L. S. J. Biol. Chem., 1972, 247: 5034-5040.

Freeze Drying

The cotton sprouts of 4 days were collected and frozen in liquidnitrogen and freeze dried (Hile et al. 1972). After this process, thematerial was crushed in a pestle and freed from fibrous material andsettled up to obtain a pretty homogeneous meal of about 0.5 mesh.

Acetonic Powders Preparation

The meals were defatted using n-hexane (1:10 w/v) at room temperatureand then treated with acetone at −20° C. to remove pigments andpolyphenolic compounds, in this case mainly gossypol. The powdersobtained were stored in a dry box at room temperature.

Carboxypeptidase Assay

The acetonic powder (30 mg) was suspended in buffer (4 ml) and incubatedat 37° C. for 30, 60, 90, 120 minutes. The proteolysis was stoppedadding 1 ml of 12% trichloroacetic acid. In this case, the buffer usedwas 0.1 M phosphate pH 6.5 (Hile et al. 1972). The control was asuspension of the same sample prepared in the same way but, in thiscase, it was immediately deactivated, adding 1 ml of trichloroaceticacid. The trials with treated and untreated samples were carried out atthe same time.

Photometric Analysis

Proteins were precipitated with trichloroacetic acid and then removed byfiltration, before with Whatman paper n. 4 and then by micro-filtration(Orange Scientific Braine I'Alleud, Belgium (Ø0.2 μm)). Finally, thehydrolyzed protein samples were analyzed as clear solutions bydetermining the absorbance at 280 nm in a quartz cuvette.

Each series of analysis was performed twice. The comparison between thevalues of carboxypeptidase activity of treated and untreated samples wasdone using the average value of each series of analysis.

From the data shown in FIG. 4 it may be inferred that the proteolyticactivity related to carboxypeptidase in treated samples is lower than inthe untreated ones.

In Table 3 the percentage of these differences is also reported.

TABLE 3 Carboxypeptidase activity of treated and untreated cottonsprouts at the 4^(th) day of germination. Untreated Cotton Sprouts PBOTreated Cotton Sprouts Exp. n° Activity Activity Time 1 2 Mean (U) 1 2Mean (U) Δ U % 0 0.759 0.695 0.727 0.632 0.729 0.68 — 30′ 0.894 0.8580.876 0.149 0.730 0.763 0.746 0.066 0.083 55.7 60′ 0.925 0.942 0.9330.206 0.752 0.858 0.805 0.125 0.081 39.3 90′ 0.988 0.956 0.927 0.2000.880 0.838 0.859 0.113 0.087 43.5 120′ 0.943 1.030 0.986 0.259 0.7750.787 0.781 0.101 0.158 61.0Endopeptidase Assay

The acetonic powder (30 mg) was suspended in buffer (4 ml) and incubatedat 37° C. for 15′, 30, 60, 90, 120 minutes. The proteolysis was stoppedadding 1 ml of 12% trichloroacetic acid. In this case the buffer usedwas 0.1 M phosphate pH 7.7 (Funkhouser et al. 1980). The control was asuspension of the same sample prepared in the same way but wasimmediately deactivated, adding up 1 ml of trichloroacetic acid. Thetrials with treated and untreated samples were carried out at the sametime.

Photometric Analysis

Proteins were precipitated with trichloroacetic acid and then removed byfiltration, before with Whatman paper n. 4 and then by micro-filtration(Orange Scientific Braine I'Alleud, Belgium (Ø0.2 μm)). Finally, thehydrolyzed protein samples were analyzed as clear solutions bydetermining the absorbance at 280 nm in a quartz cuvette.

Each series of analysis was performed threefold. The comparison betweenthe values of endopeptidase activity of treated and untreated sampleswas done using the average value of each series of analysis. As it ispossible to see in FIG. 5, also the endopeptidase activity in PBOtreated cotton sprouts is lower than in the untreated ones. In Table 4the percentage of these differences is also reported.

TABLE 4 Endopeptidase activity in PBO treated and untreated cottonsprouts at 4 days of germination. Untreated Cotton Sprouts CottonSprouts Treated with PBO Activity Activity Time 1 2 3 Mean (U × 10³) 1 23 Mean (U × 10³) Δ U % 0 850.00 845.00 847.50 == 821.00 897.00 886.00868.00 == == == 15′ 953.00 956.00 925.00 944.67 97.17 965.00 876.00915.00 918.67 50.67 46.50 47.86 30′ 972.00 965.00 952.00 963.00 115.50965.00 935.00 950.00 950.00 82.00 33.50 29.00 60′ 1008.00 1014.001030.00 1017.33 169.83 1038.00 1038.00 972.00 1016.00 148.00 21.83 12.8690′ 1031.00 1045.00 1003.00 1026.33 178.83 1015.00 991.00 980.00 995.33127.33 51.50 28.80 120′ 1021.00 985.00 1037.00 1014.33 166.83 1017.00960.00 1015.00 997.33 129.33 37.50 22.48

Example 4 Effect of PBO on Bt-Toxin Content in Transgenic Cotton PlantSamples

With the aim to find the correlation between Bt toxin levels and theeffect of the PBO treatment on cotton plants, the Bt toxin concentrationwas measured in Bt transgenic cotton plant samples (cv.SicalaV2i)treated with PBO or untreated (the control).

The assay was carried out on samples collected at different plantdevelopment stages, which were: 4, 35, 60, 85 days after sowing.

Materials and Methods.

In order to determine the Bt toxin concentration in PBO treated oruntreated cotton samples, a specific ELISA assay was used. The ELISACry1Ab/Cry1Ac Plate Kit was obtained from Envirologix (Portland,Maine—USA). The assay was performed according to the Envirologixprotocol with the following modifications:

-   1. The cotton freeze-dried powder got from leaf or cotyledon tissues    was used instead of the fresh and whole leaf tissue.-   2. The Bt toxin extraction from the cotton freeze-dried powder was    done with a sample/buffer ratio of 1:25 (w/v).-   3. The extraction was made leaving the sample in the buffer for at    least 4 hours at room temperature, instead of using the Envirologix    Disposable Tissue Extractor. The extract was then clarified by    centrifugation at 5,000×g for 5 min at room temperature.

FIG. 6 a shows the Bt concentrations in PBO treated or untreated cottonplant samples at the plant development stages reported above. In FIG. 6and in table is also shown that there is a significant lower Bt contentin PBO-treated samples at 35, 60, 85 days after planting, whereas nodifference was found at 4 days. FIG. 6 b shows the difference inpercentage between PBO-treated and untreated cotton plant samples.

In table 5 are summarized the results obtained with the described assay.

TABLE 5 Bt content and differences between PBO treated with anduntreated samples in transgenic cotton Cry1Ac ppm Growth stage cv V2i cvV2i PBO Δ: treated − %: treated − days (tissue) untreated treatedcontrol control 4 (plantlets) 46.86 44.60 −2.26 −4.823 35 (cotyledons)60.99 66.57 5.58 9.149 35 (leaves) 32.85 45.45 12.6 38.356 60 (leaves)26.64 31.07 4.43 16.629 85 (leaves) 28.18 31.91 3.73 13.236 Δ:difference in values treated minus control (cv V2i untreated)

Example 5 Effect of PBO Treatment on Protease (Endoprotease) Activity inCotton Plants at Different Growing Stages

With the aim to evaluate the action of PBO on the proteolytic activityin cotton plants, the enzymatic activity was tested on samples of PBOtreated and untreated cotton leaves (as control). The samples werecollected at different development stages of plants, which were: 4,0.35, 60 and 85 days after sowing. These samples were also analysed fordetermining the Bt content (as described in example 4). To this aim, theenzymatic activity was tested on crude freeze dried leaf extracts withtwo different assays: the Radial Diffusion Assay (RDA), in which aprotein, such as the bovine gelatine, was used as a substrate to testthe endopeptidase activity, and a photometric assay described in thenext example, in which DL-BAPA was used as a substrate, to determine ageneral peptidase activity.

Materials and Methods

Plant Culture

Cotton seeds of the cv. Sicala V2i were previously rinsed in distilledwater and then placed in equal number in suitable plastic boxescontaining settled and sterile sand. Seeds were watered with equalvolumes of distilled water and the culture boxes were then covered witha plastic film to avoid evaporation during germination, which wascarried out in the dark for 4 days at 30° C. The seeds to be analysedafter 4 days of germination were watered with distilled water or with anequal volume of a micro-suspension saturated of PBO (2% w/v). After 4days, these sprouts were collected for testing, whereas the othersprouts were moved in suitable pots and located in the greenhouse at 24°C. during the day and 20° C. in the night.

Treatments

At fixed times of 4, 35, 60, 85 days after sowing, 10 plants fairlyhomogeneous for height were treated with a commercial PBO formulation(Endura) 2% (w/v). Treatments were given out vaporizing the dilutedformulation on the plants taking care to treat both leaves sides.

Freeze Drying

Two days after each treatment, a sample of the treated plants, and thecorrespondent controls, were collected and all leaves were frozen inliquid nitrogen and freeze dried. After this process, the material waspowdered in a pestle and then stored in a dry box at room temperature.

Extract Preparation

A powder aliquot, which represents each step of plant growth, of thetreated and controls samples was extracted in two steps. The powdersample was extracted with buffer 0.1 M Tris-HCl pH 7.7 (1:20 w/v) usingan Ultraturrax blender at 24,000×RPM for 2 min in a ice bath to avoidoverheating. The extract was then clarified by centrifugation at 5,000×gat 10° C., for 20 min. The clear supernatant was collected and filtratedwith Whatman paper no 4. The pellet was further extracted with the sameprocedure, with the exception that in this case the extraction ratio was1:10 (w/v). Finally, the supernatants of the two extractions processeswere joined and then concentrated 4-5 times by centrifugation at 3,000×gat 15° C. for 2 hours, using suitable concentrators (Amicon Inc.Beverly, Mass.—USA), fitted with a membrane of 10 κD cut off.

Protein Concentration

For each extract, the amount of soluble proteins was determined by theBiuret method and the enzymatic activity was then expressed as specificactivity i.e. Units/mg soluble proteins.

RDA was performed as described in Santarius, K. and Ryan, C., Anal.Biochem., 1977, 77: 1-9 in Petri dishes containing bovine gelatine assubstrate, dispersed in agar gel. Each dish contained 5 wells of which 4were filled with suitable aliquots of concentrated cotton extracts,whereas one was filled with a bovine β-trypsin as standard solution (0.2U/ml). The dishes were covered and sealed with a plastic film to avoidevaporation and then incubated at 37° C. for 16 hours.

The enzymatic activity in the protein-agar gel produced a clear radialdiffusion zone around each circular wells, due to the bovine gelatineproteolysis against an opaque background.

The trypsin equivalents were calculated by measuring the radii of theclear zones produced by the plant extracts and compared to a standardcurve obtained by a serial dilution of trypsin. Each dish contained astandard solution of trypsin that was used to plot a standard curve foreach plate, thus the endopeptidase activity was expressed as units oftrypsin equivalents.

Each analysis was performed in quadruplicate. FIG. 7 shows the values ofendopeptidase activity obtained with RDA of the treated and untreatedcotton samples. This experiment points out that the commercial PBOformulation (Endura), especially at 35, 60, 85 days after sowing,significantly inhibited the endopeptidase activity in cotton leaves ofSicala V2i genotype. In table 6 are shown the differences betweentreated and untreated cotton samples as percentages.

TABLE 6 Endopeptidase activity of PBO treated and untreated samples bythe RDA assay. U/mg protein Difference % Days after cv. V2i Bt cv. V2iBt Treated minus sowing untreated PBO untreated 4 5.36 5.90 9.15 35 8.478.04 −5.35 60 5.67 4.07 −39.31 85 4.82 4.14 −16.42 U = trypsinequivalents

The photometric assay was carried out as described in Erlanger, B. F. etal. Arch. Biochem., 1961, 95: 271-278. In the assay is measured theabsorbance variation due to DL-BAPA hydrolysis at 410 nm. The assay wasperformed mixing directly in the cuvette 20 μl of cotton crude extractwith 980 μl of DL-BAPA in buffer 0.1 M Tris-HCl pH 8.2. One peptidaseunit was defined as the amount of the enzyme which produces one unit ofabsorbance variation (410 nm) per minute at pH 8.2 and 30° C.

Each series of analysis was performed in duplicate or triplicate. FIG. 8reports the values of peptidase activity in PBO treated and untreatedcotton samples calculated as U/mg of soluble protein. In this figure isshown a maximum of peptidase activity around 60 days of development. Atthis time the treatment with commercial PBO formulation (Endura) shows amaximum significant inhibition of the proteolytic activity on the SicalaV2i cotton. Differences in the proteolytic activity are observedthroughout the plant growing cycle by this assay in PBO treated vsuntreated cotton plants. A statistically significant inhibition isobserved during the most part of the plant growing cycle. Finally, intable 7 is shown the specific activities of peptidase of treated anduntreated Sicala V2i cotton samples. In this table it is also reportedthe activity differences in percentage between the treated and untreatedsamples.

TABLE 7 Peptidase activity (U/mg of soluble protein) of PBO treated anduntreated samples, in the photometric assay with DL-BAPA as substrate.U/mg of soluble protein × 10⁻² Difference % Days after untreated PBOtreated Treated minus sowing cv. V2i Bt cv. V2i Bt untreated 4 4.95 5.214.99 35 11.65 9.43 −23.54 60 17.10 13.62 −25.55 85 10.10 12.42 18.68

1. A method consisting of treating plants with a benzodioxole derivativeof formula I as the only active ingredient

wherein R₁, R₂ and R₃, either the same or different, are selected fromthe group consisting of: hydrogen; alkyl C₂-C₄; and CH₂OR₄ where R₄ isselected from the group consisting of: hydrogen, and —(CH₂CH₂O)_(n)—R₅,in which n is an integer from 1 to 2 and R₅ is selected from the groupconsisting of: hydrogen, alkyl C₁-C₄, and benzyl, and where, when R₁, R₂and R₃ are all the same, they can never be hydrogen, for modulating theactivity or the content of proteolytic enzymes in plants transgenic forat least one Bacillus thuringiensis toxin.
 2. The method according toclaim 1 wherein R₁, R₂ and R₃ either the same or different are selectedfrom the group consisting of: hydrogen, propyl, and CH₂OR₄ where R₄ is—(CH₂CH₂O)₂—R₅ and wherein in R₅ said alkyl is a C₂-C₄.
 3. The methodaccording to claim 2 wherein the benzodioxole derivative has formula II:


4. The method according to claim 1 wherein said proteolytic enzymes areselected from the group consisting of: carboxypeptidases, andendopeptidases.
 5. The method according to claim 4 wherein saidendopeptidases are selected from the group consisting of: serineproteases, cystein proteases, cathepsins, and metallo-endopeptidases. 6.The method according to claim 5 wherein said cystein proteases areselected from the group consisting of: bromelain, calpain, ficin,papain, and chymopapain.
 7. The method according to claim 1 wherein saidplants transgenic for at least one Bacillus thuringiensis toxin areselected from the group consisting of: soya, maize and cotton.
 8. Themethod according to claim 1 wherein the at least one Bacillusthuringiensis toxin is a Bacillus thuringiensis Cry protein.
 9. Themethod according to claim 8 wherein the Cry protein is selected from thegroup consisting of: CryI, Cry II, Cry III, and Cry IV.
 10. The methodaccording to claim 9 wherein said protein is the CryIA protein ofsubtype (a), (b) or (c).
 11. The method according to claim 1 whereinsaid modulation is a negative modulation.
 12. The method according toclaim 1 wherein said benzodioxole derivatives are mixed with suitableemulsifiers.
 13. The method according to claim 12 wherein saidemulsifiers are selected from the group consisting of: calcium salts ofalkylarylsulfonic acids, polyglycol esters of fatty acids,alkylarylpolyglycol ethers, polyglycol ethers of fatty alcohols,condensation products of ethylene oxide-propylene oxide, alkylpolyethers, esters of sorbitanic fatty acid, esters ofpolyoxyethylenesorbitanic fatty acid, and sorbitanic esters ofpolyoxyethylene.
 14. The method according to claim 13 wherein saidemulsifiers are selected from: alkylarylpolyglycol ethers and calciumsalts of alkylarylsulfonic acids.
 15. The method according to claim 1wherein the treating is performed with benzodioxole derivative offormula II:

at a concentration from 50 to 800 grams/hectare.
 16. The methodaccording to claim 15 wherein said concentration is from 100 to 400grams/hectare.
 17. The method according to claim 16 wherein the treatingis repeated up to three times per vegetative cycle.
 18. The methodaccording to claim 17 wherein at least one treating is performed at theend of the vegetative cycle.
 19. The method according to claim 15wherein said treating is carried out by nebulisation.
 20. The methodaccording to claim 16 wherein said concentration is from 200 to 350grams/hectare.
 21. The method according to claim 19 wherein the treatingis concentrated on the air-exposed areas of the plant.